Extracellular matrix vesicles are believed to play a key role in the initial stages of calcification in cartilage, bone and dentin, but their mechanism of action is still not well understood. The objective of the proposed research is to elucidate the mechanism of endochondral calcification with special emphasis on the role of matrix vesicles. Attention will be focused on 1) determining factors involved in the formation and release of matrix vesicles by cells, 2) continued characterization of their enzymes, proteins, mineral forms and membrane constituents, and 3) further study of the sequences and stoichiometry of Pi and Ca metabolism by matrix vesicles in the induction of mineral formation. In particular, studies will be aimed at a) eludicating the role of calcium phosphate-induced membrane fusion, and of acidic phospholipid-Ca-Pi complexes, in the formation of matrix vesicles, b) ascertaining the requirement of preloaded mineral in subsequent matrix vesicle mineralization, and c) determining the role of alkaline phosphatase, and other vesicle proteins, in the transport and accumulation of Pi and Ca during matrix vesicle-mediated mineralization. We will continue to use epiphyseal cartilage from long bones of rapidly growing chickens as our experimental tissue. This source provides abundant, actively calcifying material for isolation of cells and matrix vesicles, and studying their metabolism. Techniques used in this research project will be tissue culture, cell fractionation, 45Ca- and 32Pi-metabolism, chemical and physical methods: chromatography (column, thin-layer, HPLC, 2-dimensional, etc.), electrophoresis (PAGE, SDS-PAGE, isoelectric focusing), enzymolegy (purification, kinetic analyses), spectroscopy (UV, IR, NMR, MAS-NMR), electron microscopy (transmission, scanning, freeze-fracture), x-ray diffraction, etc.